Novel uses of parapoxvirus preparations

ABSTRACT

The present invention related to use of Parapoxvirus preparations for the treatment of conditions related to infections with strictly intracellular bacteria

FIELD OF THE INVENTION

The present invention relates to use of Parapoxvirus preparations forthe treatment of conditions related to infections with Chlamydia andother strictly intracellular bacteria.

BACKGROUND OF THE INVENTION

It is known from several in vivo studies that the prophylacticapplication of Parapoxvirus preparations can strengthen the immuneresponse. This therapeutic efficacy has been applied to infections withvirus as well as bacteria in animal health. For example, the use ofParapoxvirus preparations in bovine mastitis due to Staphylococcusaureus infection showed a significant reduction of infection (Zecconi etal., 1999). Likewise, immunostimulation of leukocytes have beendescribed in animals as well as humans (Yirrell et al., 1994; Haig etal., 1996; Haig et al., 1999). The main mechanism of the stimulationdetected is the induction of different cytokines (e.g., interferons α orγ, diverse interleukins). In summary, the published results showed thatParapoxvirus preparations stimulates cells of the immune system byincreasing the amount of soluble mediators.

BAYPAMUN®, a pharmaceutical product, is used to induce “paraspecificimmunity,” i.e., for inducing the unspecific immune system. It is usedtherapeutically, metaphylactically, and prophylactically for thetreatment of animals in need. BAYPAMUN® is manufactured from chemicallyinactivated Parapoxvirus ovis strain D1701 (German Patent DE3504940).The inactivated Parapoxvirus ovis induces in animals non-specificprotection against infections with a wide variety of extracellularpathogens. It is assumed that this protection is mediated via variousmechanisms in the body's own defense system. These mechanisms includethe induction of interferons, the activation of natural killer cells,the induction of “colony-stimulating activity” (CSA), apoptosis, and thestimulation of lymphocyte proliferation. Earlier investigations of themechanism of action demonstrated the stimulation of interleukin-2 andinterferon-α.

New Zealand patent application No. 512341 (filed Jul. 13, 2001)discloses that individual viral proteins of Parapoxvirus ovis stain NZ2and groups of NZ2 proteins can mimic the effect of preparation of thefull virus particle.

Also known is that Parapoxvirus preparations can be used to treatinfections with extracellular bacteria. What is not previously known isthat Parapoxvirus preparations can be used to treat infections withstrictly intracellular bacteria, such as Chlamydia. This finding issurprising and unexpected since the human or animal body's defensemechanisms against strictly intracellular bacteria is very differentfrom the body's defense mechanism against extracellular pathogens andbacteria.

The human and animal immune system protects the human or animal bodyfrom invasion of microbial cells. The so-called first-line-of-defenseare cells of the innate immune system, mainly granulocytes andmacrophages. Both cell types attack and phagocytose microbial pathogensdirectly. However, they need further support from the specific immunesystem. T-cells and B-cells are the most prominent members of theso-called specific immune response. B-cells produce specific antibodieswhich specifically detect, opsonize and neutralize microbial invaders.T-cells are the major players for the production and release of specificsoluble factors, which activate other immune effector cells. Anotherspecific function of T-cells is the killing of infected or otherwiseabnormal cells in the body (e.g., tumors). The mechanism of antibodyopsonization and direct attack cannot work for strictly intracellularbacteria as they are not floating freely in the blood. Due to theirintracellular nature, these pathogens can be indirectly attacked by theimmune system. In this context, the T-cells mediate an effective immuneresponse (e.g., via the secretion of specific mediators likeinterferons). This process lead to an activation of the cells of theinnate immune system, enabling them to overcome the intracellularpathogens by killing them intracellularly. On the other hand, theT-cells recognize the infected cells and mediate lysis or cell death.Due to release of the pathogens from their intracellular habitat, theyare thereafter accessible to different defense mechanisms.

Intracellular bacteria can be divided into 2 main groups: firstly, thefacultative intracellular bacteria (like Listeria, Mycobacteria,Salmonella, and Legionella) able to grow also outside the eukaryotichost cell, and secondly, strictly intracellular species (such as, e.g.,Chlamydia spec., Chlamydia pneumoniae, Chlamydia psittaci, Chlamydiatrachomatis) strictly dependent on intracellular growth. Allintracellular pathogens are also known to infect domestic animals,thereby mediating different pathologies. Intracellular bacteria are ingeneral difficult in therapy.

Due to the nature of strictly intracellular bacteria and theirrespective habitat (e.g., phagosome, cytoplasm), antibiotics are verylimited in the ability to achieve resolution of an infection. Theantibiotic used in therapy must enter the host cell and remain active.Due to the chemical nature of some antibiotics, this is achieved andthese antibiotics (e.g., quinolones, macrolides) are used in infectionswith intracellular pathogens, although with limited efficacy. Thepathology of intracellular bacteria is mainly a chronic one, leading toeven more difficult therapeutic efficacy.

It is known to a person skilled in the art that conditions, such aspneumonia (Grayston et al., 1989), arteriosclerosis (Leinonen, 2000),multiple sclerosis (Moses and Sriram, 2001), arthritis (Inman et al.,2000), and asthma (Cook, 1999) can all be related to infections withstrictly intracellular bacteria.

Hence, it is desirable to have available methods or pharmaceuticalcompositions to prophylactically or therapeutically treat infectionswith strictly intracellular bacteria and diseases and conditions relatedto these infections in humans as well as in non-human animals.

SUMMARY OF THE INVENTION

The present invention relates to the use of Parapoxvirus preparationsfor the prophylaxis or therapy of infections with strictly intracellularbacteria. In a preferred embodiment, the invention relates to the use ofParapoxviruses preparations (e.g., preparations of Parapoxvirus ovis,Parapoxvirus ovis strain D1701, Parapoxvirus ovis strain NZ2,Parapoxvirus ovis strain NZ7, Parapoxvirus ovis strain NZ10, orf virus,or orf-11 virus) for the prophylaxis or treatment of infections withstrictly intracellular bacteria. The present invention also relates topharmaceutical compositions and methods of manufacture of pharmaceuticalcompositions, comprising Parapoxvirus preparations, useful in thetreatment of infections with strictly intracellular bacteria.

In a preferred embodiment of the invention, the strictly intracellularbacterial infection to be treated is an infection with Chlamydia. In amore preferred embodiment of the invention, the strictly intracellularbacterial infection to be treated is an infection with Chlamydiapneumoniae, Chlamydia psittaci, and/or Chlamydia trachomatis. Infectionswith Chlamydia are known to lead to other diseases and pathologicalstates, such as, e.g., pneumonia, arteriosclerosis, arteriosclerosis,arthritis, asthma, and other diseases. It is well known to personsskilled in the art that conditions, such as pneumonia (Grayston et al.,1989), arteriosclerosis (Leinonen, 2000), multiple sclerosis (Moses andSriram, 2001), arthritis (Inman et al., 2000), and asthma (Cook, 1999)can all be related to infections with strictly intracellular bacteria.One embodiment of the invention relates to the treatment of thesediseases and pharmaceutical compositions for use in the treatment orprophylaxis of these diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows lung weights of mice 12 days after intranasal infectionwith 2×10⁶ IFU Chlamydia psittaci B577 and treatment with BAYPAMUN® inthe early phase of the infection. Error bars indicate the standard errorof the mean. The lung weight increase of infected mice over that ofnaive, unchallenged mice is a direct measure of disease intensity atthis time after inoculation.

DETAILED DESCRIPTION OF THE INVENTION

“Conditions related to infection with strictly intracellular bacteria,”within the meaning of the invention, are diseases and conditions whichare related to or caused by infections with strictly intracellularbacteria. Such conditions, within the meaning of the invention are,e.g., but not limited to, pneumonia, arteriosclerosis, multiplesclerosis, arthritis, and asthma. Infection with strictly intracellularbacteria itself is also regarded to be “a condition related to infectionwith strictly intracellular bacteria.”

A “protein,” within the meaning of the invention, is any polypeptide ofat least five amino acids.

“Recombinant Parapoxvirus protein,” within the meaning of the invention,is any protein encoded by a Parapoxvirus genome, that is expressed by orin a cell, to which cell the coding polynucleotide was introduced usingrecombinant DNA technology. Recombinant DNA technology encompasses theuse of bacterial vectors, viral vectors, other vectors, DNA molecules,and other agents for transferring nucleic acids into a host cell. Othertechniques of recombinant DNA technology relating to the invention, suchas electroporation, the use of competent cells, and other techniques arewell known to a person skilled in the art.

“Parapoxvirus preparations,” within the meaning of the invention, isunderstood as being any biological material which is obtained from, orpresent in, any member of the Parapoxvirus genus. In a preferredembodiment of the invention, Parapoxvirus preparations also compriserecombinant proteins encoded by a Parapoxvirus genome. These recombinantproteins can be used alone or in any combination.

Parapoxvirus preparations can contain biological material obtained fromor coded by the genome of more than one member of the Parapoxvirusgenus. In a preferred embodiment of the invention, Parapoxviruspreparations also comprise suitable carriers and/or adjuvants and othersubstances that are useful in the preparation of a pharmaceuticalcomposition. In a preferred embodiment of the invention, theParapoxvirus preparation contains only inactivated biological material.

New Zealand patent application No. 512341 (filed Jul. 13, 2001)discloses that individual viral proteins of Parapoxvirus ovis stain NZ2and groups of NZ2 proteins can mimic the effect of preparation of thefull virus particle. Likewise, Parapoxvirus proteins or recombinantParapoxvirus proteins are effective individually and in combination withother Parapoxvirus proteins or recombinant Parapoxvirus proteins for thetreatment of conditions related to infection with strictly intracellularbacteria.

German Patent DE3504940 (published on Nov. 9, 1997) contains a detaileddescription of methods for the manufacture of Parapoxvirus preparations.Other methods for the manufacture of Parapoxvirus preparations are knownto a person skilled in the art.

The present invention relates to the use of Parapoxvirus preparationsfor the prophylaxis or therapy of infections with strictly intracellularbacteria and conditions related to such infections. In a preferredembodiment of the invention, the invention relates to the use ofParapoxvirus preparations comprising Parapoxvirus material fromParapoxviruses (such as, e.g., Parapoxvirus ovis, Parapoxvirus ovisstrain D1701, Parapoxvirus ovis strain NZ2, Parapoxvirus ovis strainNZ7, Parapoxvirus ovis strain NZ10, orf virus, orf-11 virus) for theprophylaxis or treatment of infections with strictly intracellularbacteria.

In a preferred embodiment of the invention, the Parapoxvirus preparationcontains only inactivated biological material. Parapoxvirus material canbe inactivated by methods well known to a person skilled in the art,such as, e.g., by radiation.

In a preferred embodiment of the invention, the strictly intracellularbacterial infection to be treated is an infection with Chlamydia.Infections with Chlamydia are related to other diseases and pathologicalstates, such as, e.g., but not limited to, pneumonia, arteriosclerosis,multiple sclerosis, arthritis, and asthma. One embodiment of theinvention relates to the treatment of these diseases and topharmaceutical compositions for use in the treatment or prophylaxis ofthese diseases.

Parapoxvirus and recombinant Parapoxvirus proteins can be administeredsystemically (e.g., intravenously (i.v.), subcutaneously,intramuscularly, intracutaneously, intraperitoneally), locally, ororally (per os). The recombinant proteins or products thereof should beformulated appropriately, e.g. in a non-pyrogenic solution or suspensionfor i.v. use, or in capsules for implantation, or in capsules for per osuse. Pharmaceutical compositions of the invention can be administered,e.g., oral, nasal, anal, vaginal etc., as well as parenteraladministration. Pharmaceutical compositions of the invention can be inthe form of suspensions, solutions, syrups, elixirs, or appropriateformulations in polymers as well as liposomes.

The invention also relates to recombinant Parapoxvirus proteins.Recombinant Parapoxvirus proteins of the invention can be prepared,e.g., with suitable recombinant cell lines. Alternatively,non-recombinant cell lines, such as WI-38, MRC-5, or Vero cells, can beinfected with recombinant viruses that carry the recombinant genes usingviral vectors such as, but not limited to, the Vaccina virus (e.g.,Vaccina lister). In addition, other suitable viruses can be used incombination with other suitable cells (e.g., using Vaccinia virusvectors and fibroblasts as host cells or baculovirus vectors and insectcells as host cells). It is advantageous to cultivate the recombinantcell cultures in high-cell-density fermentations to achieve favorableproductivity and a good overall process performance.

The present invention relates to the use of Parapoxvirus preparationsfor the manufacture of a pharmaceutical composition for the prophylaxisor for the treatment of conditions related to infection with strictlyintracellular bacteria.

In a preferred embodiment, the invention further relates to the use ofParapoxvirus preparations for the manufacture of a pharmaceuticalcomposition for the prophylaxis or for the treatment of conditionsrelated to infection with strictly intracellular bacteria, wherein theinfection is Chlamydia.

In a preferred embodiment, the invention further relates to the use ofParapoxvirus preparations for the manufacture of a pharmaceuticalcomposition for the prophylaxis or for the treatment of conditionsrelated to infection with strictly intracellular bacteria, wherein theParapoxvirus preparation comprises material from a Parapoxvirus ovis, orfrom a Parapoxvirus ovis strain NZ2, or from a Parapoxvirus ovis strainNZ7, or from a Parapoxvirus ovis strain NZ10, or from a Parapoxvirusovis strain D1701, or from an orf virus, or an orf-11 virus.

In a preferred embodiment, the invention further relates to the use ofParapoxvirus preparations for the manufacture of a pharmaceuticalcomposition for the prophylaxis or for the treatment of conditionsrelated to infection with strictly intracellular bacteria, wherein thecondition related to infection with strictly intracellular bacteria isarteriosclerosis, and/or pneumonia, and/or multiple sclerosis, and/orasthma, and/or arthritis.

In a preferred embodiment, the invention further relates to the use ofParapoxvirus preparations for the manufacture of a pharmaceuticalcomposition for the prophylaxis or for the treatment of conditionsrelated to infection with strictly intracellular bacteria, wherein theParapoxvirus preparation comprises recombinant Parapoxvirus protein.

The invention further relates to a method of treatment of conditionsrelated to infections with strictly intracellular bacteria byadministering to a subject in need a therapeutically effective dose of aParapoxvirus preparation.

In a preferred embodiment, the invention further relates to a method oftreatment of conditions related to infections with strictlyintracellular bacteria by administering to a subject in need atherapeutically effective dose of a Parapoxvirus preparation, whereinthe infection is Chlamydia.

In a preferred embodiment, the invention further relates to a method oftreatment of conditions related to infections with strictlyintracellular bacteria by administering to a subject in need atherapeutically effective dose of a Parapoxvirus preparation, whereinthe Parapoxvirus preparation comprises material from Parapoxvirus ovis,or from a Parapoxvirus ovis strain NZ2, or from a Parapoxvirus ovisstrain NZ7, or from a Parapoxvirus ovis strain NZ10, or from aParapoxvirus ovis strain D1701, or from an orf virus, or an orf-11virus.

In a preferred embodiment, the invention further relates to a method oftreatment of conditions related to infections with strictlyintracellular bacteria by administering to a subject in need atherapeutically effective dose of a Parapoxvirus preparation, whereinthe condition related to infection with strictly intracellular bacteriais arteriosclerosis, and/or pneumonia, and/or multiple sclerosis, and/orasthma, and/or arthritis.

In a preferred embodiment, the invention further relates to a method oftreatment of conditions related to infections with strictlyintracellular bacteria by administering to a subject in need atherapeutically effective dose of a Parapoxvirus preparation, whereinthe Parapoxvirus preparation comprises recombinant Parapoxvirus protein.

The invention further relates to a pharmaceutical composition for use inthe treatment of prophylaxis of conditions related to infections withstrictly intracellular bacteria, wherein said pharmaceutical compositioncomprises a Parapoxvirus preparation.

In a preferred embodiment, the invention further relates to apharmaceutical composition for use in the treatment of prophylaxis ofconditions related to infections with strictly intracellular bacteria,wherein said pharmaceutical composition comprises a Parapoxviruspreparation, wherein the infection is Chlamydia.

In a preferred embodiment, the invention further relates to apharmaceutical composition for use in the treatment of prophylaxis ofconditions related to infections with strictly intracellular bacteria,wherein the Parapoxvirus preparation comprises material from aParapoxvirus ovis, or a Parapoxvirus ovis strain NZ2, or a Parapoxvirusovis strain NZ7, or a Parapoxvirus ovis strain NZ10, or a Parapoxvirusovis strain D1701, or an orf virus, or an orf-11 virus.

In a preferred embodiment, the invention further relates to apharmaceutical composition for use in the treatment of prophylaxis ofconditions related to infections with strictly intracellular bacteria,wherein the condition related to infection with strictly intracellularbacteria is arteriosclerosis, and/or pneumonia, and/or multiplesclerosis, and/or asthma, and/or arthritis.

In a preferred embodiment, the invention further relates to apharmaceutical composition for use in the treatment of prophylaxis ofconditions related to infections with strictly intracellular bacteria,wherein the Parapoxvirus preparation comprises recombinant Parapoxvirusprotein.

EXAMPLES Example 1

The effect of Parapoxvirus on the response of mice to challenges withChlamydia psittaci (C. psittaci) B577 was investigated.

Mice of the group “live C. psittaci B577 vaccinated, challenged” servedas controls for optimum protection and received a low-level intranasalinfection with 3×10⁴ inclusion forming units (IFU) of C. psittaci in 20microliters sucrose-phosphate-glutamate (SPG) buffer. This infectiontypically confers complete resistance to subsequent homologous challengein BALB/c mice.

Four weeks later, all groups except “naive, non-challenged” werechallenged intranasally with a high dose of 3×10⁶ IFU C. psittaci B577.This is approx. equivalent to an LD₂₀ 12 days after inoculation. LD₂₀ isthe dose that leads to the death of approx. 20% of the test animals.

BAYPAMUN® treated groups received 3 intraperitoneal injections: 16 hoursprior to challenge, 48 hours later, and 96 hours later, each 100microliters of BAYPAMUN® dissolved in H₂O, or further diluted in PBS 1:6or 1:36 (“Baypamun undiluted, challenged,” “Baypamun diluted 1:6,challenged,” “Baypamun diluted 1:36, challenged,” respectively).

At the maximum of the early disease (inflammatory) response on day 4after inoculation, mice of group “naive, challenged” were very scruffyand clinically sick, while the “live C. psittaci B577 vaccinated,challenged” group appeared healthy, and BAYPAMUN®-treated groups weresomewhat scruffy, but much less so that the “naive, challenged” group.

After day 4, all groups except group “naive, challenged” recoveredquickly and appeared clinically healthy, while mice of group “naive,challenged” appeared progressively emaciated and developed pumpingrespiration. Three mice in this group were sacrificed on days 10 and 11after inoculation (p.i.) prior to the scheduled date on day 12 p.i.,since they would not have survived.

On day 12 p.i., all mice were sacrificed. The low lung weight and lowlung weight increase (measure of disease intensity) of the group “liveC. psittaci B577 vaccinated, challenged” reflects the typical protectiveimmune response and a lung without macroscopic lesions and microscopicinterstitial in-filtrate, but with prominent, microscopicperibronchiolar lymphocytic cuffs (FIG. 1). Mice of group “Baypamun 1:36diluted, challenged” were essentially identical to group “live C.psittaci B577 vaccinated, challenged” with the exception of one mousewhich had a lung with visible tissue consolidation and increased weight.Mice of the other BAYPAMUN® treated groups had bimodal responses, withsome mice without any macroscopic lesions while others had clear areasof tissue consolidation due to interstitial pneumonia. However, theselesions generally were less severe than in the severely diseased “naive,challenged” group.

In conclusion, it was shown that Parapoxvirus has a dramatic protectiveeffect against infections with strictly intracellular bacteria,especially with Chlamydia.

REFERENCES

-   German patent application DE3504940-   Cook, 1999. Antimicrobial therapy for Chlamydia pneumoniae: its    potential role in atherosclerosis and asthma. J. Antimicrob.    Chemother., 44(2):145-8.-   Grayston et al., 1989. Current knowledge on Chlamydia pneumoniae,    strain TWAR, an important cause of pneumonia and other acute    respiratory diseases. Eur J Clin Microbiol Infect Dis.,    8(3):191-202.-   Haig and Fleming, 1999. Vet. Immunol. Immunopathol. 72: 81-86.-   Haig et al., 1996. Vet. Immunol. Immunopathol. 54:261-267.-   Inman et al., 2000. Chlamydia and associated arthritis. Curr Opin    Rheumatol., 12(4):254-62.-   Leinonen, 2000. Chlamydia pneumoniae and other risk factors for    atherosclerosis. J Infect Dis., 181 Suppl 3:S414-416.-   Moses and Sriram, 2001. An infectious basis for multiple sclerosis:    perspectives on the role of Chlamydia pneumoniae and other agents.    BioDrugs. 15(3):199-206.-   Raupach and Kaufmann, 2001. Curr. Opin. Immunol. 13: 417-428.-   Yirrel et al., 1994. British J. Dermatol. 130: 438-443.-   Zecconi et al., 1999. Efficacy of a biological response modifier in    preventing Staphylococcus aureus intramammary infections after    calving. J. Dairy Sci., 82(10):2101-2107

1. Use of Parapoxvirus preparations for the manufacture of apharmaceutical composition for the prophylaxis or for the treatment ofconditions related to infection with strictly intracellular bacteria. 2.Use of claim 1, wherein the infection is with Chlamydia.
 3. Use of claim1, wherein the Parapoxvirus preparation comprises material from aParapoxvirus ovis, or from a Parapoxvirus ovis strain NZ2, or from aParapoxvirus ovis strain NZ7, or from a Parapoxvirus ovis strain NZ10,or from a Parapoxvirus ovis strain D1701, or from an orf virus, or anorf-11 virus.
 4. Use of claim 1, wherein the condition related toinfection with strictly intracellular bacteria is arteriosclerosis,and/or pneumonia, and/or multiple sclerosis, and/or asthma, and/orarthritis.
 5. Use of claim 1, wherein the Parapoxvirus preparationcomprises recombinant Parapoxvirus protein.
 6. Method of treatment ofconditions related to infections with strictly intracellular bacteria byadministering to a subject in need a therapeutically effective dose of aParapoxvirus preparation.
 7. Method of claim 6, wherein the infection iswith Chlamydia.
 8. Method of claim 6, wherein the Parapoxviruspreparation comprises material from Parapoxvirus ovis, or from aParapoxvirus ovis strain NZ2, or from a Parapoxvirus ovis strain NZ7, orfrom a Parapoxvirus ovis strain NZ10, or from a Parapoxvirus ovis strainD1701, or from an orf virus, or an orf-11 virus.
 9. Method of claim 6,wherein the condition related to infection with strictly intracellularbacteria is arteriosclerosis, and/or pneumonia, and/or multiplesclerosis, and/or asthma, and/or arthritis.
 10. Method of claim 6,wherein the Parapoxvirus preparation comprises recombinant Parapoxvirusprotein.
 11. Pharmaceutical composition for use in the treatment ofprophylaxis of conditions related to infections with strictlyintracellular bacteria, wherein said pharmaceutical compositioncomprises a Parapoxvirus preparation.
 12. Pharmaceutical composition ofclaim 11, wherein the infection is with Chlamydia.
 13. Pharmaceuticalcomposition of claim 11, wherein the Parapoxvirus preparation comprisesmaterial from a Parapoxvirus ovis, or a Parapoxvirus ovis strain NZ2, ora Parapoxvirus ovis strain NZ7, or a Parapoxvirus ovis strain NZ10, or aParapoxvirus ovis strain D1701, or an orf virus, or an orf-11 virus. 14.Pharmaceutical composition of claim 11, wherein the condition related toinfection with strictly intracellular bacteria is arteriosclerosis,and/or pneumonia, and/or multiple sclerosis, and/or asthma, and/orarthritis.
 15. Pharmaceutical composition of claim 11, wherein theParapoxvirus preparation comprises recombinant Parapoxvirus protein.